PROJECTSUMMARY The aggregation and accumulation of alpha-synuclein (aSyn) fibrils results in histopathological inclusions calledLewybodiesandLewyneuritesandisobservedinseveralneurodegenerativedisorders.InParkinson?s disease(PD),thisculminatesinthedegenerationofdopaminergicneuronsinthesubstantianigraandsevere motor impairment. Emerging findings suggest that PD may have a prodromal phase characterized by non- motorsymptomssuchasgastrointestinaldisturbances.Thus,itishypothesizedthatpathologicaSynmightfirst accumulate in peripheral tissue before propagating to the brain where it precipitates PD pathology. This is supported by research that shows that aSyn fibrils are capable of interneuronal transport and can seed the formation of additional fibrils from endogenous aSyn. The goal of this proposal is to provide me with training in advanced techniques to interrogate peripheral nervous systems in mice. I will use these techniquestotestthehypothesisthatpathologicaSynpropagatesfromentericneuronsinthegutto the brain via the vagus nerve in an age- and activity-dependent manner, eventually damaging the dopaminergicsystemthatcoordinatesmovement.TovisualizeaSynfibrils,entericpopulations,andnerve tractsinhighresolution,IwillusetheCLARITYtissueclearingmethoddevelopedinourlabthatrenderswhole organs and organisms optically transparent and macromolecule permeable. I will also use novel quantitative measures to analyze large three-dimensional datasets. I will use a novel adeno associated virus capsid that hasincreasedaffinityforperipheralneuronstodeliverconstructstoneuronsandnervesintheentericnervous system. I will use whole cell patch clamp recordings to determine the effect of aSyn fibrils on enteric neuron electrophysiology and neurotransmission. Lastly, I will use optogenetics to explore an activity-dependent mechanism of aSyn uptake and release. Upon completion, the experiments detailed in this proposal will contributetoourunderstandingofpathologicaSynformationinthegutandtheirpropagationtothebrain,and willbekeyindevelopingnoveldiagnosticandtherapeuticstrategiesforPD.